Transplantation experiments by Spemann and Mangold (1924, Arch. Mikroskopische Anat. Entwicklungsmechanik, 100:599-638) established the presence of an anatomically discrete region, the Spemann organizer, or dorsal lip, that controls patterning of the developing body axis in vertebrate embryos. Diffusible factors emanating from this region were found to be involved in different developmental processes. The arrangement of Drosophila cuticle hairs in a defined polarity was found to be genetically controlled by ‘frizzled’ (FZD1), a 7-transmembrane receptor with a large extracellular cysteine-rich domain.
The bovine and human homologs of FZD1 were cloned by RT-PCR and by screening bovine articular cartilage and human placenta cDNA libraries, which identified cDNAs encoding FRZB, the mammalian analog of FZD1 (Hoang et al., 1996, J. Biol. Chem., 271:26131-26137). The deduced 325-amino acid bovine and human FRZB proteins share 94% amino acid identity. Sequence analysis predicted that FRZB contains a 25-amino acid signal peptide, an N-terminal N-glycosylation site, a 24-amino acid putative transmembrane segment, a region with multiple potential Ser/Thr phosphorylation sites, and a serine-rich C-terminal domain. The N-terminal region of FRZB shares 50% amino acid identity, including the conservation of 10 Cys residues, with frizzled. Immunoblot analysis determined that FRZB is expressed as an approximately 36-kD protein. In situ hybridization analysis of human embryos representing different stages of development detected no expression from week 6 through week 13 except in the developing appendicular skeleton, as well as in several craniofacial bones and epiphyseal ends of the rib cage. Immunochemical analysis confirmed the expression of FRZB in the developing skeletal structures.
Northern blot analysis revealed that FRZB is expressed strongly in placenta and heart, at intermediate levels in brain, skeletal muscle, kidney, and pancreas, and at low levels in lung and liver (Leyns et al., 1997, Cell 88:747-756). SDS-PAGE analysis detected secretion of FRZB, possibly after proteolytic cleavage, consistent with FRZB's lack of the 7 transmembrane domains found in the Drosophila and vertebrate frizzled gene family. Functional analysis in Xenopus embryos showed that FRZB can antagonize the early and late effects of WNT8 signaling. Mammalian WNT genes include oncogenes that lead to mammary tumors. For further characterization of FRZB, see, e.g., Dann et al., 2001, Nature 412:86-90; Rattner et al., 1997, Proc. Nat. Acad. Sci. 94:2859-2863; and Schumann et al., 2000, Cardiovasc Res. 45:720-728.
Leyns et al. (supra) mapped the human FRZB gene to 2q31-q33. They noted that loss of one copy of the 2q arm occurs with high incidence in lung and colorectal carcinomas, as well as in neuroblastomas, and suggested that FRZB might function as a tumor suppressor gene. Hoang et al. (supra) suggested FRZB might play a role in skeletal morphogenesis. However, a direct role for the FRZB gene in human disease and development has not been identified.
Among other aspects, the present invention provides alleles of FRZB, identified by the presence of one or more predisposing or protective polymorphisms, that are associated with an increased or decreased risk for obesity and/or osteoporosis. A complete understanding of the invention will be obtained upon review of the following.